We have explored the minimum sequence requirement for the initiation of apolipoprotein B (apoB)-mediated triglyceride-rich lipoprotein assembly. A series of apoB COOH-terminal truncation mutants, spanning a range from apoB34 (amino acid residues 1-1544 of apoB100) to apoB19 (residues 1-862) were transfected into COS cells with and without coexpression of the microsomal triglyceride transfer protein (MTP). ApoB34, -25, -23, -21, -20.5, and -20.1 underwent efficient conversion to buoyant lipoproteins when coexpressed with MTP. ApoB19.5 (amino acids 1-884) also directed MTP-dependent particle assembly, although at reduced efficiency. When apoB19.5 was truncated by another 22 amino acids to form apoB19, MTP-dependent lipoprotein assembly was abolished. Analysis of the lipid stoichiometry of secreted lipoproteins revealed that all apoB truncation mutants formed spherical particles containing a hydrophobic core. Even highly truncated assembly-competent forms of apoB, such as apoB19.5 and 20.1, formed lipoproteins with surface:core lipid ratios of <1. We conclude that the translation of the first ϳ884 amino acids of apoB completes a domain capable of initiating nascent lipoprotein assembly. The composition of lipids recruited into lipoproteins by this initiating domain is consistent with formation of small emulsion particles, perhaps by simultaneous desorption of both polar and neutral lipids from a saturated bilayer.

The phylogenic dissection of MTP and apolipoprotein B function, coupled with ongoing structural and biochemical analyses, provide significant insights into the mechanisms of lipid mobilization and secretion. Some of these factors and processes may be targeted therapeutically to modulate the quantitative and qualitative aspects of apolipoprotein B production.

To better understand the earliest steps in the assembly of triglyceride (TG)-rich lipoproteins, we compared the biophysical and interfacial properties of two closely related apolipoprotein B (apoB) truncation mutants, one of which contains the complete lipoprotein initiating domain (apoB20.1; residues 1-912), and one of which, by virtue of a 50 amino acid C-terminal truncation, is incapable of forming nascent lipoproteins (apoB19; residues 1-862). Spectroscopic studies detected no major differences in secondary structure, and only minor differences in conformation and thermodynamic stability, between the two truncation mutants. Monolayer studies revealed that both apoB19 and apoB20.1 bound to and penetrated egg phosphatidylcholine (EPC) monolayers; however, the interfacial exclusion pressure of apoB20.1 was higher than apoB19 (25.1 mN/m vs. 22.8 mN/m). Oil drop tensiometry revealed that both proteins bound rapidly to the hydrophobic triolein/water interface, reducing interfacial tension by ?20 mN/m. However, when triolein drops were first coated with phospholipids (PL), apoB20.1 bound with faster kinetics than apoB19 and also displayed greater interfacial elasticity (26.9 6 0.8 mN/m vs. 22.9 6 0.8 mN/m). These data establish that the transition of apoB to assembly competence is accompanied by increases in surface activity and elasticity, but not by significant changes in global structure. Apolipoprotein B (apoB) is a 4,536 amino acid residue secretory glycoprotein that serves as the major structural component of triglyceride (TG)-rich lipoproteins secreted by the liver (very low density lipoproteins) and intestine (chylomicrons) (1-5). The assembly of apoB-containing lipoproteins is believed to occur in two stages: in the first stage, a precursor apoB-containing emulsion particle is formed in the endoplasmic reticulum (ER) concurrently with apoB translation; in the second stage, these nascent lipoproteins fuse with TG-rich emulsion particles produced in the smooth ER (6-11). Both steps require the activity of microsomal TG transfer protein (MTP), a dedicated ER-localized cofactor that is essential for apoBcontaining lipoprotein assembly and secretion (12)(13)(14).The mechanism by which apoB is lipidated during the initial phase of TG-rich lipoprotein assembly is not well understood. It was first proposed that apoB intercalates into the inner leaflet of the ER membrane during its translation and then entrains membrane lipid as it desorbs from the ER membrane (15-18). The observation that apoB becomes associated with the ER membrane immediately after translation (16), and that N-terminal fragments of apoB bind to hydrophobic surfaces (19)(20)(21)(22)(23)(24), suggests that apoB possesses avid surface activity, which is an essential requisite of this model. Recently, however, have proposed a different model for the lipidation of apoB that is based upon sequence similarities between apoB and vitellogenin. This model postulates that the N terminal ?1,000 residues of apoB form two b-sheets that comprise the s...

The amino-terminal 20.1% of apolipoprotein B (apoB20.1; residues 1-912) is sufficient to initiate and direct the formation of nascent apoB-containing lipoprotein particles. To investigate the mechanism of initial lipid acquisition by apoB, we examined the lipid binding and interfacial properties of a carboxyl-terminal His 6 -tagged form of apoB20.1 (apoB20.1H). ApoB20.1H was expressed in Sf9 cells and purified by nickel affinity chromatography. ApoB20.1H was produced in a folded state as characterized by formation of intramolecular disulfide bonds and resistance to chemical reduction. Dynamic light scattering in physiological buffer indicated that purified apoB20.1H formed multimers, which were readily dissociable upon the addition of nonionic detergent (0.1% Triton X-100). ApoB20.1H was incapable of binding dimyristoylphosphatidylcholine multilamellar vesicles, unless its multimeric structure was first disrupted by guanidine hydrochloride. However, apoB20.1H multimers spontaneously dissociated and bound to the interface of naked and phospholipid-coated triolein droplets. These data reveal that the initiating domain of apoB contains solvent-accessible hydrophobic sequences, which, in the absence of a hydrophobic lipid interface or detergent, engage in self-association. The high affinity of apoB20.1H for neutral lipid is consistent with the membrane binding and desorption model of apoB-containing lipoprotein assembly. ApoB3 is the major structural protein of hepatic very low density lipoproteins and intestinal chylomicrons (1, 2). The assembly of apoBcontaining lipoproteins proceeds in two steps: cotranslational incorporation of the nascent apoB polypeptide into a precursor lipoprotein particle in the rough ER followed by the additional posttranslational lipidation via fusion with lipid droplets in the ER and/or the Golgi (3-6). Both steps of apoB-containing lipoprotein assembly require MTP, a dedicated cofactor composed of a unique 97-kDa subunit complexed with the ubiquitous ER-localized folding enzyme, protein-disulfide isomerase (7,8). During the first step of lipoprotein assembly, MTP may transfer lipid molecules from the ER membrane or other donor sites directly to apoB and/or may bind to apoB and promote its proper folding and autonomous lipid acquisition (7, 9). MTP also participates in bulk transmembrane lipid transport from the cytosol into the ER-Golgi, a process critical for second step apoB particle expansion (10 -12).Several models of the first initiating step of lipoprotein assembly have been proposed. The earliest and most commonly accepted theory posits that the apoB polypeptide interacts with the inner leaflet of the ER membrane during translation, causing the nucleation and desorption of an ϳ20-nm diameter emulsion particle upon the completion of translation (13-17). A variation of this model proposes that the interfacial binding and lipoprotein desorption process is completed upon translation of only the first ϳ1000 amino-terminal residues of apoB, giving rise to a small, high density lipoprot...